The Effect of Oryza Sativa L. Subsp. Japonica Cultivar Yukihikari on The Immune System

Oryza sativa L. subsp. japonica cultivar Yuhikikari is proposed to alleviate atopic dermatitis, although its mechanism is unclear. To clarify this issue, we evaluated the effect of Yukihikari on the immune system in vitro and in vivo by comparison with another Japanese cultivar Kirara397. A DNA microarray analysis of mouse spleen cells cultured with either Yukihikari or Kirara397 showed that Yukihikari-added spleen cells exhibited signicantly reduced expression of pro-inammatory genes. Furthermore, another transcriptome analysis of the cultivars by RNA sequencing showed a similar result to the DNA microarray analysis. Mice fed with Yukihikari had less germinal center B cells, fewer autoantibodies, and less weight than those fed with Kirara397. These results suggest that Yukihikari has immune-regulatory functions and accounts for its allergy-ameliorating effects.


Introduction
Rice (Oryza sativa L.) is the most popular main staple worldwide. A large number of cultivars have been generated by breeding to improve their agronomic and economic characteristics, such as high yield and superior eating quality.
It has been shown that main staples, including rice and wheat, contain allergenic proteins. These proteins were rst reported in rice by Shibasaki et al. to be in the albumin/globulin fractions of rice endosperm proteins 1 . Allergens in rice have been identi ed 2,3 and hypoallergenic rice has been genetically or enzymatically generated [4][5][6] . In addition to eggs, milk, and wheat, rice allergy is a common food allergy in Japan. In Hokkaido, Japan, the rice cultivar transition from Yukihikari to Kirara397 due to eating quality has been implicated as a reason for the increase in atopic dermatitis. Indeed, the reversal of rice consumption from Kirara397 to Yukihikari alleviated atopic dermatitis. Furthermore, it has been suggested that in an animal model Yukihikari modulated gut microbiota, and augmented gut barrier function 7 . However, there is almost no difference in the allergenic protein contents of these cultivars.
Although the anti-allergic effect of Yukihikari on atopic dermatitis has been postulated, its mechanism is still unclear.
The bene cial effect of food on our health has been intensively studied. Probiotics, including lactic acid bacteria (LAB), modulate the immune system 8,9 . To analyze their functions, we established various assay systems and methodologies, including intravital imaging of the gut 8,10-15 , transcriptome analyses of immune cells 9 , and generation of genetically-engineered model mice 15 . Using these technologies, we recently found that LAB derived from miso-induced interleukin 22 (IL-22) production in immune cells, thereby resulting in an augmentation of the skin barrier function 8 .
In this study, the function of Yukihikari on the immune system was elucidated, and we applied our established protocols to Yukihikari.lso, distinct immunological functions of Yukihikari from that of Kirara397were highlighted. harvested, bulked, air-dried, and dehulled. By sieving, brown rice grains smaller than 1.9 mm were removed. The remaining were polished using a rice-polishing machine (TOYO TESTER Seimaiki MC-90A, Toyo Rice Co., Ltd.) until 90% of the grains by weight were white. After which rice powder was prepared from the polished white rice.

Cells and mice
Spleen cells of C57BL/6 mice were prepared as previously described 9 .
In vitro immunological assay A total of 1 × 10 6 spleen cells were cultured in 1-mL RPMI1640 medium containing 10% fetal calf serum (FCS) with or without 10 μg of the rice powder for 2 d. The activation cell surface markers-CD69 and CD86-on spleen cells were evaluated by ow cytometry.

Measurement of the anti-DNA antibody levels
Immunoglobulin levels were measured as previously described 9 using enzyme-linked immunosorbent assays (ELISAs). Sonicated herring sperm DNA (Sigma-Aldrich, 10 µg/ml) was coated on ELISA plates. The following antibodies were used: alkaline phosphatase-conjugated anti-IgM and anti-IgG (Southern Biotech).

Transcriptome analyses
A total of 4 × 10 6 spleen cells were cultured in 4-mL RPMI1640 medium containing 10% FCS with 20 μg of rice powder for 2 d. Total RNA was prepared from spleen cells using ISOGEN II (Nippon Gene). Gene expression analysis was performed using DNA microarray and RNA sequencing.
For DNA microarray analysis, the measurement was entrusted to Macrogen Japan. Microgen provided the method as follows. Microarray results were extracted using the feature extraction software v11.0 (Agilent Technologies). Raw data for a similar gene were then summarized automatically using the Agilent feature extraction protocol to generate a raw data text le, providing expression data for each gene probed on the array. The array probes with Flag A in samples were ltered out. The selected gProcessedSignal (gpS) value was transformed logarithmically and normalized using the quantile method. Statistical signi cant difference of expression data was determined using fold change. For DEG set, the hierarchical cluster analysis was performed using complete linkage and Euclidean distance as a measure of similarity. Gene-enrichment and functional annotation analysis for the signi cant probe list was conducted using gene ontology (www.geneontology.org/). Data analyses and visualizations of differentially expressed genes were conducted using R v.3.5.1 (www.r-project.org). The SurePrint G3 Mouse Gene Expression 8x60K microarray kit was used for DNA microarray analysis (Agilent Technologies). Finally, data were analyzed using the genetic manifested software R v.2.15.1.
For RNA sequencing analysis, total RNAs were subjected to RNA sequencing analysis (GENEWIZ). RNA-Seq library construction, next-gen sequencing and following bioinformatics analyses were conducted by GENEWIZ. Brie y, total RNA was quanti ed and quali ed by Qubit RNA Assay (Invitrogen), and RNA ScreenTape (TapeStation; Agilent Technologies). Poly(A) mRNA was enriched using magnet beadsconjugated oligo(dT) and following library preparation for high-throughput sequencing was conducted according to the manufacturer's procedure (NEBNext Ultra II RNA Library Prep Kit for Illumina; New England BioLabs). Approximately 250 ng of total RNA was used as initial input for mRNA selection and adapter-ligated double stranded cDNA fragment was ampli ed 12 cycles PCR, which incorporates Illumina P5/P7 adapters and sample-speci c barcode sequence. Fragment size and quantity of libraries established were con rmed by Qubit DNA Assay (Invitrogen) and DNA ScreenTape (TapeStation; Agilent Technologies). Libraries with unique sample barcodes were pooled together and loaded onto an Illumina HiSeq/NovaSeq instrument according to manufacturer's instructions (Illumina). Sequencing was carried out using 150 bp paired-end (PE) con guration. Image analysis, base calling and demultiplex were conducted by the Illumina standard software. Approximately 20M PE reads (6 Gb output in 150 bp PE con guration) per samples were obtained. The raw sequencing reads were ltered to remove adapter and low quality reads. The resulting clean reads were used for mapping to the reference genome (Mus musculus; Ensembl/GRCm38), quantifying gene expression, studying differential gene expression and further downstream analyses.

Statistical analysis
Experimental data are presented as the mean ± standard deviations (S.D). Statistically signi cant differences were evaluated using a two-tailed Student's t-test for unpaired data. P values <0.05 were considered to be statistically signi cant difference was considered as P < 0.05.

Data availability
The data that support the ndings of this study are available from the corresponding author, T. A., upon reasonable request.

Results
Yukihikari and Kirara397 did not alter cell-surface expression of T/B cell activation markers on spleen cell in vitro.
We established an in vitro screening method for probiotics based on the activation markers-CD69 on T cells and CD86 on B cells-and found some LAB possessing immune-modulatory functions 9 . To elucidate the effect of Yukihikari on the immune system, Yukihikari and Kirara397 were subjected to this screening method. The addition of these cultivars did not alter cell populations, such as B, T, and dendritic cells ( Supplementary Fig. 1). Furthermore, both cultivars did not upregulate the activation markers, CD69 and CD86 on B cells as well as CD69 on T cells, suggesting that both cultivars lack salient immunestimulatory functions.

Yukihikari induced immunoregulatory gene expression in spleen cells in vitro
To nd these differences, we analyzed the gene expression pro les of mouse spleen cells cultured with either Yukihikari or Kirara397 using DNA microarray. Compared with Kirara397, 562 and 586 genes were signi cantly up-and down-regulated by Yukihikari ( Supplementary Fig. 2). Among them, the immune response and in ammatory gene expression levels of Yukihikari-treated cells were signi cantly lower than those of Kirara397-treated cells (Table 1). Gene expressions of the pro-in ammatory cytokines-IL-1, IL-6, and TNF-a were lower in Yukihikari-treated cells than those of Kirara397, suggesting that Yukihikari possesses anti-in ammatory functions (Fig. 1).
Also, further transcriptome analysis using RNA sequencing of spleen cells cultured with cultivars Yukihikari and Kirara397 was performed ( Supplementary Fig. 3). RNA-sequencing analysis of Yukihikariand Kirara397-treated cells exhibited substantially similar results to that of the DNA microarray analysis ( Table 2). Yukihikari down-regulated activation markers-CD69 and CD86-at mRNA levels although cellsurface protein levels were not altered (Figs. 2 and 3). Gene expression of anergy markers in B cells, such as ApoE and CD72 16 were upregulated in spleen cells by Yukihikari. Furthermore, FoxP3, a master regulator of regulatory T cells, 17 was upregulated by this cultivar. Also, inhibitory receptors, such as CD22, CD72, Fc receptor for IgG2b (FcgR2b), and PIR-B 18,19 were upregulated. In contrast, proin ammatory cytokines, such as IL-6, IL-1b, and TNF-α 20 were down-regulated. Additionally, the GC B cell marker, B cell lymphoma 6 (Bcl-6) gene was down-regulated by Yukihikari. These results strongly suggest that Yukihikari has immune-suppressive functions.
At the protein level, the expression of FoxP3 in spleen cells cultured with rice powder for 2 d was con rmed. FoxP3 was slightly higher in cells treated with Yukihikari than those treated with Kirara397 (Fig.4). Inhibitory receptors on spleen cells were almost similar (data not shown).
To validate the effect of Yukihikari on the immune system in vivo, mice were fed either Yukihikari or Kirara397 for 6 weeks and their immune cell populations in peripheral lymphatic tissues, such as the spleen, PP, and MLN were analyzed (Fig. 5). Cell surface markers, CD4, CD8, B220, and CD11c were stained as helper T cells, cytotoxic T cells, B cells, and dendritic cell markers, respectively, although other minor cell populations expressed these markers. In the spleen and PP, these populations were insigni cantly different in the two groups of mice. However, the B cell ratio in MLN of the Yukihikari-fed mice decreased. Additionally, GC B cells (GL-7 + B220 + cells), follicular helper T (Tfh) cells (PD-1 + ICOS + cells or PD-1 + CXCR5 + cells), and regulatory T (Treg) cells (FoxP3 + CD4 + cells) in these mice were analyzed. Tfh and Treg cells were almost similar in the two groups of mice, whereas GC B cells in PP and MLN were less similar in the Yukihikari-fed mice.
We generated IgA de cient (IgA -/-) mice, which exhibited in ammation in the small intestine 21 . First, we examined whether IgA -/mice generated autoantibodies or not. Compared with the wild-type mice, IgA -/mice produced anti-DNA antibodies ( Fig.6a and b). As anti-DNA IgG production in IgA -/mice, IgA -/augmented autoimmunity. Furthermore, anti-DNA antibodies were measured after feeding in IgA -/mice to evaluate the immune-suppressive effect of Yukihikari on autoimmunity. Serum anti-DNA antibodies in Yukihikari-fed mice were lower than that of Kirara397-fed mice ( Fig.6c and d). These results suggest that Yukihikari negatively regulates B cell activation in vivo.
Also, an increase in the bodyweight of Yukihikari-fed mice was slower than that of Kirara397-fed mice ( Fig.6e and f).

Discussion
To clarify the effect of Oryza sativa L. subsp. japonica cultivar Yuhikikari on the immune system, we analyzed its functions based on our protocol, which was recently established 8,9 . Transcriptome analyses using DNA microarray and RNA sequencing revealed that Yukihikari possesses immune-suppressive and anti-in ammatory functions. In contrast, Kirara397 possesses a slight immune-stimulatory function. Yukihikari elevated Treg in vitro and likely augmented it in vivo. Yukihikari-fed mice exhibited less autoantibody and less weight.
In this study, for the rst time, transcriptome analyses showed that Yukihikari has a distinct immunological function from Kirara397. Yukihikari upregulated the inhibitory receptors-CD22, CD72, FcgR2b, and PIR-B-on B cells 18,19 , although the upregulation of the Cd72 gene is marginal. These receptors negatively regulated signaling of the B cell antigen receptor (BCR) 18,19 . Additionally, ApoE was upregulated by Yukihikari. Interestingly, CD72 and ApoE have been identi ed as molecules of the upregulated gene in anergic B cells 16 , suggesting that Yukihikari negatively affected B cells. Following this notion, activation marker genes on B cells such as Cd86, Cd69, and C40 were down-modulated by Yukihikari. Also, notably, FoxP3-a master regulator of Treg 17 -was upregulated by Yukihikari. Indeed, spleen cells treated with Yukihikari increased Treg cells. Moreover, Yukihikari-fed mice tend to have more Treg cells than those of Kirara397-fed mice. Alternatively, Kirara397 down-regulated CD22, PIR-B, ApoE, and FoxP3 as well as upregulated CD69 and CD86, therefore, suggesting that it slightly augments the immune system. Additionally, in ammatory cytokines-IL-1b, IL-6, TNF-a, and IFN-γ-gene expression were more decreased by Yukihikari than Kirara397, suggesting that Yukihikari has an anti-in ammatory function.
Upon activation, conventional B2 B cells generated a secondary follicle called germinal center (GC) where class-switch and hypersomatic mutation occurs 22,23 . Bcl-6-a master regulator of the GC B cellsdecreased in spleen cells by Yukihikari, whereas it increased by Kirara397. In accordance with in vitro results, Yukihikari-fed mice exhibited more GC B cells in PPs and MLNs than that of Kirara397-fed mice, indicating that humoral immunity was suppressed by Yukihikari. Regulatory T cells-master regulators of the FoxP3 gene, which were upregulated in vitro by Yukihikari slightly increased in vivo by Yukihikari in comparison with Kirara397, although it is an insigni cant difference. Compared to the Kirara397-fed mice, serum anti-DNA antibodies, which might be involved in autoimmune diseases were more reduced in Yukihikari-fed IgA -/mice than in. These effects of Yukihikari on the immune system may justify previous clinical ndings, which stated that changing dietary rice from Yuhikikari to Kirara397 relieved atopic dermatitis.
Compared with Yukihikari-fed mice, Kirara397-fed mice increased more in body weight. Since Yukihikari possesses anti-in ammatory functions, in ammation may be reduced in Yukihikari-fed mice. This might be a reason for reduced weight because in ammation induces the anti-in ammatory hormone, cortisol, which is responsible for fat storage in visceral fat tissues 24 . Further studies will be required to clarify this mechanism.
Yukihikari modulates the gut microbiota and augments the gut barrier function 7 . However, in this study, we showed that Yukihikari negatively regulated spleen cells in vitro, it acts on the immune system directly. Since dendritic cells can capture luminal molecules through the intestinal epithelium and migrate to PPs and lymph nodes, possibly Yukihikari directly regulates the immune system in vivo in a gut microbiota independent manner.
In conclusion, Yukihikari possesses immune-suppressive and anti-in ammatory functions, which may prevent allergies and various in ammation-oriented diseases, such as lifestyle diseases and major neurocognitive disorders. Further studies are required to justify these functions and clarify their mechanisms. Tables Table 1. Gene ontology of DNA microarray analysis of "Yukihikari" and "Kirara397"-treated spleen cells.